Part:BBa_K782015

12x[NicTAL] operator_CMV promoter_mCitrine

• DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

Introduction

Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.

Our construct contain twelve specific binding sites for NicTAL12 upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm. (Figure 1). After binding of NicTAL12:KRAB con binding sites, a repression of reporter protein mCitrine occurs.

Single binding sequence for NicTAL12 is: TCTATCAATGATAGA

Figure 1. Shematic representation of twelve specific binding site for TALD:KRAB upstream of CMV promoter and reporter protein mCitrine.

Characterization

Results: 12x[NicTAL] binding sites were further characterized with other reporter constructs.

• mCitrine was provided from host lab.
• Binding sites for TAL effectors were ordered from GeneArt.

References

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 464
  Illegal XhoI site found at 1094
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 16
  Illegal AgeI site found at 248
  
• 1000
  COMPATIBLE WITH RFC[1000]